With the approval of the proteasome inhibitor Velcade for multiple myeloma therapy, the ubiquitin pathway has been validated for drug discovery. An alternative ubiquitin-associated degradation pathway is lysosomal;for example, the ubiquitin E3 ligase Cbl promotes degradation of membrane bound epithelial growth factor receptor (EGFR) by ubiquitylating the receptor, marking it for lysosomal degradation rather than recycling to the membrane. Removal of ubiquitin by an isopeptidase would spare EGFR and favor recycling and enhanced mitogenesis. This proposal is focused on AMSH, a ubiquitin isopeptidase that prevents endosomal sorting and lysosomal degradation of EGFR. Chronic pulmonary diseases, including chronic obstructive pulmonary disease (COPD), asthma, and pulmonary arterial hypertension (PAH) are characterized by airway and vascular remodeling and remain extraordinarily common illnesses. EGF and EGFR are associated with the pathobiology of chronic pulmonary diseases. The aim of this project is to develop agents active against these diseases by promoting the natural degradation of EGFR. In phase I, a high throughput screen was configured for inhibitors of AMSH activity utilizing an N-terminal ubiquitin-fused substrate reporter. A similar counterscreen was validated for the isopeptidase UBPY. In addition, a cell based assay detecting EGFR degradation was validated, completing the aims of Phase I. In phase II, first, several chemical libraries will be screened using the AMSH high throughput assay, and selected hits characterized in secondary assays for selectivity. Next, efficacy studies will be performed;the ability of the best hits to promote EGFR degradation and inhibition of EGFR activity will be evaluated using human airway smooth muscle (ASM), pulmonary arterial vascular smooth muscle (PVSM) cells and human lung fibroblasts. It will be determined whether lead compounds regulate EGFR levels in these cells and whether the effects modulate EGF-induced cell proliferation in a concentration and time-dependent manner. Concentration dependence of effects on EGF-induced activation of EGFR levels will be established by Western blot analysis. Effects of lead compounds on EGF-induced ASM, PVSM and HLFs proliferation will indicate physiological relevance. This study will provide critical information about potential efficacy of the selected leads. Finally, medicinal chemistry will be employed for the establishment of structure-activity relationships (SAR) and chemical optimization, leading to the selection of candidates for progression to preclinical development for treatment of lung disease. PUBLIC HEALTH RELEVANCE: Chronic pulmonary diseases, including chronic obstructive pulmonary disease (COPD), asthma, and pulmonary arterial hypertension (PAH) are characterized by airway and vascular remodeling and remain extraordinarily common illnesses. A prominent cellular growth factor (EGF) and its receptor (EGFR) play a role in chronic pulmonary diseases. The aim of this Phase II project is to develop agents that cause the natural degradation of EGFR in cells as a means of combating pulmonary disease. This will be accomplished indirectly by using inhibitors of a cellular enzyme, AMSH, which normally keeps cellular levels of EGFR high, thereby promoting airway inflammation and pulmonary disease. Several collections of small chemical molecules will be screened to identify inhibitors of AMSH. Screening will be accomplished using an assay developed in Phase I of this project. The most promising of these inhibitors will be tested in cellular models to see whether they act in cells to reduce EGFR levels and activity as predicted. Additional chemical modification will be performed on the best of these inhibitors to generate candidate molecules for development as drugs to treat pulmonary disease and airway inflammation.